Optical assay technique

ABSTRACT

An assay technique for qualitative and quantitative detection of a chemcial, biochemical or biological detection of a chemical, biochemical or biological species in sample. The technique comprises (a) coating at least a predetermined part of a pre-formed surface on a substrate with a thin film of a material capable of binding the species to be assayed, the pre-formed surface being optically active with respect to radiation at least over a predetermined band of wavelengths; (b) contacting the coated surface with sample; and (c) observing the optical properties of said pre-formed surface in order to determine a qualitative and quantitative change in optical properties as a result of the binding of the species onto said thin film of material. The optical properties of the pre-formed surface may be observed before and after step (b) in order to determine any change in optical properties, or they may be monitored during step (b). The pre-formed surface is preferably a grating. An article for use in the above technique is also disclosed, and comprises a substrate carrying said pre-formed surface which in turn is coated with the respective material for the species to be assayed.

This is a continuation of application Ser. No. PCT/GB83/00340, filed 21Dec. 1983.

This invention relates to an assay technique for qualitative and/orquantitative detection of chemical, biochemical or biological species ina sample.

The technique is based upon the affinity of the species which is to beassayed for a receptive material, for example a ligand or a specificbinding partner, which receptive material is coated onto a particulartype of surface.

More particularly, according to one aspect of the present invention,there is provided an assay technique for qualitative and/or quantitativedetection of a chemical, biochemical or biological species in a sample,which comprises (a) coating at least a predetermined part of apre-formed surface on a substrate with a thin film of a material capableof binding the species to be assayed, the pre-formed surface beingoptically active with respect to radiation at least over a predeterminedband of wavelengths; (b) contacting the coated surface with the sample;and (c) observing the optical properties of said pre-formed surface inorder to determine a qualitative and/or quantitative change in opticalproperties as a result of the binding of the species onto said thin filmof material.

In a first embodiment of the method of this invention, the opticalproperties of the pre-formed surface are observed before and after step(b) in order to determine any change in optical properties as a resultof the species being bound by the receptive material in the thin filmcoating on the pre-formed surface. In a second embodiment, the opticalproperties of the pre-formed surface are monitored during step (b) inorder to determine the said change in optical properties.

According to a second aspect of the present invention, there is providedan article for use in an assay technique as defined above, which articlecomprises a substrate having a pre-formed surface which is opticallyactive with respect to radiation at least over a predetermined band ofwavelengths, and at least a predetermined part of which pre-formedsurface is coated with a thin film of a material capable of binding apredetermined chemical, biochemical or biological species.

The pre-formed surface is preferably a grating. A single grating may beemployed, or the surface may comprise two or more gratings disposedmutually at an angle. Where there are two such gratings, they may bemutually orthogonal. The profile of the or each grating isadvantageously square-wave or sinusoidal. Saw-tooth profiles are alsopossible, but are not presently preferred.

The pre-formed surface may alternatively comprise a regular array ofprotuberances. With a surface of this type, and the alignment of thepeaks of the protuberances and the troughs between the protuberancescorresponds to the ridges and troughs of a grating-type structure.

The thin film of receptive material may be coated onto the pre-formedsurface so as to be deposited only in the troughs of the grating or inthe troughs between the protuberances. A monomolecular layer of thereceptive material will suffice and will generally be preferred, whetheror not the coating is confined to the troughs.

The surface structure of the pre-formed surface and in particular thedimensions of the surface relief pattern will be selected according tothe nature of the species which is to be assayed. In general, we havefound three ranges of surface depth (peak-to-trough measurements) to beadvantageous. In the first, the grating or protuberance or each of thegratings or protuberances, in the event several are employed, have adepth in the range 10 to 50 nanometers. In the second, the depth is inthe range 50 to 200 nanometers; and in the third, the depth is in therange 200 to 2000 nanometers. With the first of these ranges, the pitch(period) of the each grating(s) or the periodicity of the protuberancesis advantageously greater than their depth; the structure thuscorresponds, in general, to that of a shallow grating. With the secondand third ranges, the pitch (period) of the or each grating or theperiodicity of the protuberances is advantageously of the same order astheir depth.

In a first group of embodiments, the pre-formed surface is structured sothat it is optically active with respect to radiation whose wavelengthis in the range from 700 to 1500 nanometers. In a second group ofembodiments, the pre-formed surface is structured so that it isoptically active to radiation whose wavelength falls within the rangefrom 350 to 700 nanometers.

Conveniently, the substrate which carries the pre-formed surface isformed of a plastics material. Plastics materials curable byultra-violet light are preferred, and in particular acrylic or polyestermaterials can advantageously be used. A presently preferred plasticsmaterial is polymethylmethacrylate. A plastics substrate for use in thisinvention preferably has a refractive index in the range 1.25 to 1.6,and more preferably a refractive inded of about 1.4.

An alternative substrate is a glass coated with a synthetic polymericmaterial.

The active surface of the substrate (i.e., that surface which is, orwhich carries, the pre-formed surface) can be constituted by a metal ora metal layer. Thus a plastics substrate, e.g., ofpolymethylmethacrylate, can have adhering thereto a metal layer whichconstitutes the pre-formed surface (e.g. a single grating structure ofdepth about 250 nanometers and period about 400 nanometers). With such astructure, the plastics/metal interface may be planar, or it may conformto the surface structure of the metal layer itself. The metal used toform such layers may be gold, silver, copper or aluminium.Alternatively, the active surface of the substrate may be constituted byan inorganic oxide or a layer thereof. The inorganic oxide isadvantageously an oxide of silver, copper or aluminium. Such an oxidelayer may be produced by causing or allowing oxidation of the surface ofa metal substrate or of a metal layer adhering to a substrate of adifferent material. Where there is a layer of metal or of an inorganicoxide as just described, the layer is preferably from 5 to 50, morepreferably 10 to 30, nanometers thick.

Conveniently, the substrate is lamellar and in strip-form. Thisfacilitates use of an article in accordance with the invention incarrying out the assay.

The observation of optical properties in step (c) of the method of thisinvention can take place in transmission or in reflection. One zone ofthe pre-formed surface on the substrate may be left free of the coatingof receptive material; the method may be performed by keeping this onezone free from sample, in step (b), or by contacting the whole of thepre-formed surface, including said one zone, with the sample. Thislatter technique has advantages in that any optical effects caused bycomponents of the sample other than the species to be assayed willaffect the coated and uncoated zones equally, and thus will cancel eachother out when a comparison between the coated and non-coated zones ismade. A two-beam illuminating system can be employed in step (c) of themethod, one of the beams being directed at the uncoated zone of thepre-formed surface, and the other of the two beams being directed at apart of the coated zone of the pre-formed surface. Preferably,monochromatic radiation is used.

When the substrate and the pre-formed surface are constituted by aplastics material, and observations of the optical properties of thesurface are to be carried out in transmission, it is preferred that theuncoated, pre-formed surface when viewed in transmission normal to theplane of the surface should have a transmission not exceeding 1% for theradiation which is to be used.

In order to give optimum results when the technique of this invention isused for quantitative analysis, it may be advantageous to calibrate thecoated substrate by first carrying out the assay technique using asample containing a known proportion of the species which is to beassayed.

The present invention is applicable, for example, to testing abiological liquid, e.g. a blood sample, for specific antigen molecules.In such a case, the receptive material capable of binding the species tobe assayed will comprise antibodies for the antigen concerned.Alternatively, it is possible to use an antigen as the receptivematerial and to assay a sample for antibodies. Where the receptivematerial comprises antibodies, these are preferably monoclonalantibodies. Antigens and antibodies occur in a wide range of moleculardimensions, and the surface structure of the pre-formed surface will bedetermined in part by the size of the molecules concerned. As anexample, antigens resulting from many parasitic infections are typicallyin the size range from about 0.5 microns to 10 microns; for theseantigens, a grating pitch of greater than 6 microns and preferablygreater than 10 microns is desirable. In general, a grating pitch of theorder of twice the antigen size will be desirable.

The invention is also applicable to the assaying of other chemical,biochemical or biological species, for example ionic species. Theinvention may be used, for example, to assay the metal ion content of asample. The receptive material may be, for example, a chelating chemicalor enzyme or a chelating organism which constitutes a specific bindingpartner for the ligand or ion which is to be assayed. In general thechemical, the enzyme or organism will be one or more of: a polypeptide,a steroid, a saccharide or polysaccharide, a proteoglycan, a nucleotide,a nucleic acid, a protonucleic acid, a microbial cell or a yeast.

Application of the invention thus lies not only in the medical field fordiagnostics, but also generally in the field of process control.

The thin film of receptive material is preferably bonded firmly to thepre-formed surface of the substrate. Thus the receptive material may bebonded by electrostatic or covalent bonding to said surface.

Observations in step (c) of the method of this invention may usepolarised light. In one particular technique, the pre-formed surface ofthe substrate is in the form of a single grating of square-wave orsinusoidal profile, and the optical properties of the surface areobserved, in step (c), by monitoring the angular position at which thereoccurs a sharp reduction (dip) in reflection as the surface is observedor scanned with polarised radiation of a predetermined wavelength. Theradiation used is preferably light, and the polarisation should betransverse to the grooves of the grating.

A presently preferred article in accordance with this invention consistsof a profiled plastics strip, desirably fabricated by an embossing orcasting technique, and with a refractive index of the order of 1.4 and atransmission not exceeding 1%. The strip profile may be that of a singlegrating with square grooves, dimensioned for zero order suppression overa range of wavelengths. However, other profiles and dimensions can beused if desired, enabling diffraction efficiency into particular ordersto be enhanced or suppressed.

An article in accordance with this invention may have a plurality ofzones, each of which is coated with a different receptive material. Inthis way, a single article, e.g. in the form of a strip, can be used toassay a plurality of different species, e.g. antigens in a blood sampleor metal ions in a biochemical fluid or in an industrial effluent.

In the case of a square profile grating, if the pitch is d, the grooveheight h and the refractive index n, then zero order diffracted light ofwavelength W will be suppressed for h=W/2(n-1), whilst first orderdiffracted light will emerge at angles given by sin O=±W/d. Forapplication to blood sampling, given a grating pitch of about 6 microns,and a source wavelength of 550 nm (green), then h=0.69 microns; O=±5.2°.

The principle of the assaying method is that the receptive material,e.g. antibodies, coated on the grating are typically small molecules,e.g. sized around 10 nm, and are too small to produce any size or shapedependent light scattering. However, the antigens attached to theantibodies when a blood sample is smeared on the grating have a size ofthe same order as the wavelength of incident light, and have an effectanalogous to that of filling some of the grating grooves with water(refractive index 1.33). This means that, in the case of a gratingdimensioned as above, zero order light is no longer suppressed, whilstvery little light is diffracted into the higher orders. Generally,therefore, the transmission of the grating, normally not exceeding 1%,will be directly related to the number of antigens present.

The method thus depends on determination of the change in opticalproperties, e.g. transmission or reflection characteristics, of thegrating. For this reason, given a grating coated with antibodies overits whole area, the smearing of a part of this area with the sample canreadily enable the said change to be quantitively determined. A similareffect is preferably achieved, however, by coating only a part of thegrating with antibodies, as the antigens will not be attracted into andtrapped in the grooves in the uncoated region. Preferably, inconjunction with the last mentioned partly coated grating, a two beamilluminating system will be employed. The source may be an incandescentlamp emitting light incident on the grating through a filter. The angleof incidence of the monochromatic (or nearly monochromatic) light on thegrating is preferably 0° (i.e. normal to the grating) and, for thegrating exemplified above, zero order diffracted light would becollected by means of a lens onto a photodetector, while higher orderdiffracted light would be obscured using a stop.

One aim of the invention is to provide a low cost pre-coated gratingwhich can be widely used for diagnostic purposes, commonly in a generalpractitioner's surgery but possibly also in the home. For this purpose,the antibodies would be firmly bonded to the plastics grating, e.g. byelectrostatic bonding which can ensure virtually permanent coatingprovided that a suitable or suitably treated plastics material isinitially chosen to form the grating. As the aim would usually be todetect a specific antigen, the grating would be coated with a specificantibody, e.g. a monoclonal antibody which attracts and retains only thespecific antigen in question. Thus, successive testing of a plurality ofselectively coated gratings would enable quantitative detection ofspecific antigens as an aid to diagnosis.

The technique of smearing the grating with the sample also requiresconsideration. After wiping the grating with, say, a blood sample, it isimportant to remove any excess sample in order to ensure that minimumcarrier liquid, minimum haemoglobin and minimum large cells other thanantigen are retained.

As the effect of absorption by red cells containing haemoglobin can beminimised by suitable choice of the wavelength of illumination, it isthe retention of carrier liquid which is the next likely source oferrors of detection. For minimising such errors, the grating may bedimensioned for zero order suppression when there is a continuous liquidfilm on top of the grating; this requires a modified grating height ofh=W/2 (n1-n2), where n1 is the refractive index of the substrate and n2is the refractive index of the liquid. A liquid of high refractive indexis desirable, and one suitable example is glycerol. The smearingtechnique (i.e. step (b)) would then include the step of washing thegrating, after wiping it with the sample, with the liquid in question.

A further point to be understood in connection with the smearingtechnique is that this will commonly result in only a small percentage,e.g. less than 2%, of the overall area of the grating bearing andretaining attracted antigens. The use of a diffractor grating of highsensitivity relieves the illuminating and detector system of the extremerequirements which would otherwise be required quantitatively to detectsuch a small presence of antigen, thus making practical the use ofrelatively simple and low cost optics which can enable widespread use.

One example of assaying method and apparatus in accordance with theinvention is shown in the accompanying drawing, in which:

FIG. 1 shows an optical illuminating and detecting system;

FIG. 2 shows a detail of one embodiment of an article incorporating adiffraction grating and forming part of the system of FIG. 1;

FIG. 3 shows a cross-sectional view (not to scale) of a secondembodiment of an article incorporating a diffraction grating; and

FIG. 4 illustrates the results obtained with an article of the typeillustrated in FIG. 3. The x-axis represents the Reflectivity and they-axis represents the Angle of Incidence.

Referring first to FIG. 2, a square profile single diffraction grating10 whose pitch and depth are both equal to 800 nanometers is coated witha substantially mono-molecular layer of immobilised antibodies 12,preferably monoclonal antibodies. After smearing with a sample, anantigen 14, being a binding partner to the antibodies, is attracted andtrapped in one groove. At this point of the grating, the zero orderdiffracted light is transmitted, as indicated at 16, instead of beingsupressed.

FIG. 1 shows the grating 10 under illumination by monochromatic light18. Zero order diffracted light is collected by a lens 20 onto aphotodetector 22, while higher order diffracted light is obscured by astop 24. A two-beam illuminating system which, as described above, isgenerally preferred, will operate in a precisely analogous way.

Referring next to FIG. 3, an article in accordance with this inventionis shown in the condition after it has been contacted by a sample instep (b) of the method of the invention. The article comprises asubstrate 10 formed of polymethylmethacrylate which is about 1millimeter thick. The active (upper) surface of the substrate includes alayer 11 of aluminium of thickness 20 nanometers. This is covered by apassive film 13 of aluminium oxide (thickness one nanometer or less). Amonomolecular layer of antigen molecules 12 is covalently bonded to thefilm 13 of aluminium oxide and is thus immobilised. A layer ofantibodies 14 is attached to the antigen layer 12. This layer 14 is alsomonomolecular and is about ten nanometers thick. Isolated antigens 15have been bound by the antibodies 14. The substrate 10 with the layers11, 13, 12 and 14 constitutes one embodiment of the article of thisinvention. The pre-formed surface is in effect defined by the surface oflayer 13; this is in the form of a single grating of depth 50 nanometersand of pitch (period) 250 nanometers. The article is observed, incarrying out the method of the invention, with monochromatic light whichis polarised in a plane perpendicular to the lines of the grating; theangle of incidence of the illumination is varied and it is found thatthere is a sharp reduction (dip) in reflectivity at an angle whose valuedepends upon the amount of material (antibodies 14) overlying thearticle. The angular position of this dip, and also its angular width,are strongly dependent upon the amount of antigens attached to the layer14 of antibodies and hence these parameters provide a quantitativemeasure of the antibodies absorbed from the sample. FIG. 4 plots thereflectivity of the article against the angle of incidence of themonochromatic, polarised illumination over a small angular range. As thequantity of antigens captured by the antibody layer 14 increases, thedip in reflectivity first of all becomes more pronounced, and thenbecomes broader and the angular position of the reflectivity minimumalters, as shown in the three curves plotted. The reflectivity dip canbe considered theoretically as a plasmon resonance; it is relativelyeasy to detect a change in the angle of incidence of about 0.1 degreesor a change in the wavelength of the resonance by about 1 nanometer.Hence it is possible to detect changes corresponding to an increase inthe average thickness of the antigen layer 15 of around one nanometer.It will be appreciated that, when antigens are bound by the layer 14,the result is not the addition of a further layer of uniform thickness;nevertheless, we have found that the occurrence of isolated antigens 15over the layer 14 of antibodies behaves approximately as though theywere "smoothed out" into a layer whose average thickness modifies theoptical properties of the system as a whole.

We claim:
 1. An assay technique for qualitative and quantitativedetection of a chemical, biochemical or biological species in a sample,which comprises:(a) coating at least a predetermined part of a surfacehaving a pre-formed regular periodic surface structure provided on asubstrate with a thin film of a material capable of binding the speciesto be assayed, said surface producing a reflective dip with respect to aparameter of incident polarized light; (b) contacting the coated surfacewith the sample; (c) directing polarized light at said coated surface;and (d) measuring the change in the reflective dip as a result of thebinding of the species onto said thin film of material, whereby aquantitative measure of the species being assayed is provided.
 2. Amethod according to claim 1, wherein the reflective dip is measuredbefore and after step (b) in order to determine the said change.
 3. Amethod according to claim 1, wherein the reflective dip is monitoredwith respect to changes in the angle of incidence or wavelength of theincident polarized light.
 4. A method according to claim 1, wherein saidpre-formed regular periodic surface structure is a diffraction grating.5. A method according to claim 4, wherein the grating is of square-waveprofile.
 6. A method according to claim 4, wherein the grating is ofsinusoidal profile.
 7. A method according to claim 4, wherein thegrating is of saw-tooth profile.
 8. A method according to claim 4,wherein the grating has a depth (peak-to-trough) in the range 10 to 50nanometers.
 9. A method according to claim 8, wherein the pitch (period)of the grating is greater than its depth.
 10. A method according toclaim 4, wherein the grating has a depth (peak-to-trough) in the range50 to 200 nanometers.
 11. A method according to claim 10, wherein thepitch (period) of the grating is of the same order as its depth.
 12. Amethod according to claim 4, wherein the grating has a depth(peak-to-trough) in the range 200 to 2000 nanometers.
 13. A methodaccording to claim 1, wherein said pre-formed regular periodic surfacestructure comprises two or more diffraction gratings disposed mutuallyat an angle.
 14. A method according to claim 1, wherein said surface isstructured so that it is reflective with respect to radiation ofwavelengths from 700 to 1500 nanometers.
 15. A method according to claim1, wherein said surface is structured so that it is reflective withrespect to radiation of wavelengths from 350 to 700 nanometers.
 16. Amethod according to claim 1, wherein at least the active surface of thesubstrate is comprised of a metal or a metal layer.
 17. A methodaccording to claim 16, wherein said metal is gold, silver, copper oraluminum.
 18. A method according to claim 1, wherein the active surfaceof the substrate is comprised of an inorganic oxide or a layer thereof.19. A method according to claim 18, wherein said inorganic oxide is anoxide of silver, copper or aluminum.
 20. A method according to claim 1,wherein the substrate is in strip-form.
 21. A method according to claim1, wherein one zone of said surface distinct from said predeterminedpart of the surface on the substrate is left free of the coatingmaterial and is not contacted, in step (b), by the sample.
 22. A methodaccording to claim 21, wherein a two-beam illuminating system isemployed in step (c), one of said beams being directed at said one zoneof said surface, and the other of the two beams being directed at saidpredetermined surface part.
 23. A method according to claim 1, whereinone zone of said surface distinct from said predetermined part of thesurface is left free of the coating material and the whole of saidsurface, including said one zone, is contacted, in step (b), by thesample.
 24. A method according to claim 3 wherein the polarized light ismonochromatic and the parameter of the light monitored is its angle ofincidence.
 25. A method according to claim 1, wherein the species whichis to be detected is an antigen.
 26. A method according to claim 25,wherein the material capable of binding said species comprisesantibodies for the antigen which is to be assayed.
 27. A methodaccording to claim 26, wherein said antibodies are monoclonalantibodies.
 28. A method according to claim 11, wherein the specieswhich is to be assayed is an ionic species.
 29. A method according toclaim 28, wherein said ionic species is a metal ion.
 30. A methodaccording to claim 28, wherein the material capable of binding thespecies to be assayed is a chemical, enzyme or a organism.
 31. A methodaccording to claim 30, wherein said chemical, enzyme or organism is oneor more of: a polypeptide, a steroid, a saccharide or polysaccharide, aproteoglycan, a nucleotide, a nucleic acid, a protonucleic acid, amicrobial cell or a yeast.
 32. A method according to claim 1, whereinsaid thin film of material is firmly bonded to said surface.
 33. Amethod according to claim 1, wherein said preformed regular periodicsurface structure comprises a regular periodic array of protuberances.34. A method according to claim 33, in which said surface is washedimmediately after being contacted with the sample and before theobservations in step (c).
 35. A method according to claim 34, in whichsaid surface is covered with a layer of a liquid of high refractiveindex between steps (b) and (c).
 36. A method according to claim 1, inwhich said surface is washed immediately after being contacted with thesample and before the observations in step (d).
 37. A method accordingto claim 36, in which said surface is covered with a layer of a liquidof high refractive index between steps (b) and (c).
 38. An assaytechnique for qualitative and quantitative detection of a chemical,biochemical or biological species in a sample, which comprises:(a)coating at least a predetermined part of a surface having a pre-formedsingle diffraction grating or two or more gratings disposed mutually atan angle provided on a substrate with a thin film of a material capableof binding the species to be assayed, said surface suppressing thetransmission of zero-order diffracted light at least over apredetermined band of wavelengths; (b) contacting the coated surfacewith the sample; and (c) measuring the change in transmission ofzero-order diffracted light as a result of the binding of the speciesonto said thin film of material, whereby a quantitative measure of thespecies being assayed is provided.
 39. A method according to claim 38,wherein the transmission of said surface is measured before and afterstep (b) in order to determine the said change in transmission.
 40. Amethod according to claim 38, wherein the transmission of said surfaceis monitored during step (b) in order to determine the said change intransmission.
 41. A method according to claim 38, wherein the grating isof square-wave profile.
 42. A method according to claim 38, wherein thegrating is of sinusoidal profile.
 43. A method according to claim 38,wherein the grating is of saw-tooth profile.
 44. A method according toclaim 38, wherein the grating has a depth (peak-to-trough) in the range10 to 50 nanometers.
 45. A method according to claim 44, wherein thepitch (period) of each grating is greater than their depth.
 46. A methodaccording to claim 38, wherein the grating has a depth (peak-to-trough)in the range 50 to 200 nanometers.
 47. A method according to claim 46,wherein the pitch (period) of each grating is of the same order as theirdepth.
 48. A method according to claim 38, wherein the grating has adepth (peak-to-trough) in the range 200 to 2000 nanometers.
 49. A methodaccording to claim 38, wherein the substrate is formed of a plasticmaterial.
 50. A method according to claim 49, wherein said plasticmaterial is a material which is curable by ultra-violet light.
 51. Amethod according to claim 49, wherein said plastic material is anacrylic or a polyester material.
 52. A method according to claim 51,wherein said plastic material is polymethylmethacrylate.
 53. A methodaccording to claim 49, wherein the plastic material has a refractiveindex in the range 1.25 to 1.6.
 54. A method according to claim 53wherein the refractive index of said plastic material is about 1.4. 55.A method according to claim 38, wherein the substrate is a glass coatedwith a synthetic polymeric material.
 56. A method according to claim 38,wherein the active surface of the substrate is constituted by aninorganic oxide or a layer thereof.
 57. A method according to claim 38,wherein the substrate is in strip-form.
 58. A method according to claim38 wherein one zone of said surface distinct from said predeterminedpart of the surface on the substrate is left free of the coatingmaterial and is not contacted, in step (b), by the sample.
 59. A methodaccording to claim 58, wherein a two-beam illuminating system isemployed in step (c), one of said beams being directed at said one zoneof said surface, and the other of the two beams being directed at saidpredetermined surface part.
 60. A method according to claim 38, whereinone zone of said surface distinct from said predetermined part of thesurface is left free of the coating material and the whole of saidsurface, including said one zone, is contacted, in step (b), by thesample.
 61. A method according to claim 38, wherein in step (c)monochromatic radiation is used.
 62. A method according to claim 38,wherein the species which is to be detected is an antigen.
 63. A methodaccording to claim 62, wherein the material capable of binding saidspecies comprises antibodies for the antigen which is to be assayed. 64.A method according to claim 63, wherein said antibodies are monoclonalantibodies.
 65. A method according to claim 38, wherein the specieswhich is to be assayed is an ionic species.
 66. A method according toclaim 65, wherein said ionic species is a metal ion.
 67. A methodaccording to claim 65, wherein the material capable of binding thespecies to be assayed is a chemical, enzyme or a organism.
 68. A methodaccording to claim 67, wherein said chemical, enzyme or organism is oneor more of: a polypeptide, a steroid, a saccharide or polysaccharide, aproteoglycan, a nucleotide, a nucleic acid, a protonucleic acid, amicrobial cell or a yeast.
 69. A method according to claim 38, whereinsaid thin film of material is firmly bonded to said surface.
 70. Anarticle for use in an assay technique for qualitative and quantitativedetection of a chemical, biochemical or biological species in a sample,which comprises a surface with a regular periodic pre-formed surfacestructure reflective of polarized light at least over a pre-determinedband of wavelengths, and said surface including a metal layer capable ofsupporting surface plasmon resonance, at least a pre-determined part ofsaid surface including the metal layer being coated with a thin film ofa material capable of binding the species to be assayed.
 71. An articleas claimed in claim 70, wherein the surface is lamellar.
 72. An articleas claimed in claim 24, wherein the surface is in strip-form.
 73. Anarticle as claimed in claim 70, wherein the preformed surface structureis a diffraction grating of square-wave, sinusoidal or saw-toothprofile.
 74. An article as claimed in claim 70, wherein the preformedsurface structure comprises a regular periodic array of protuberances.75. An article as claimed in claim 70, wherein said metal is gold,silver, copper or aluminum.
 76. An article as claimed in claim 70,wherein said surface is constituted by an inorganic oxide.
 77. Anarticle as claimed in claim 76, wherein said oxide is an oxide ofsilver, copper or aluminum.
 78. An article as claimed in claim 70,wherein said thin film of material comprises antibodies.
 79. An articleas claimed in claim 70, wherein said thin film of material comprises achemical enzyme or an organism.
 80. An article as claimed in claim 70,wherein said surface is constituted by a layer at least 5 nanometers inthickness.
 81. An article as claimed in claim 70, wherein the articleincludes a plurality of zones each of which is coated with a differentreceptive material so that the article is capable of binding a pluralityof different species.
 82. An assay technique for qualitative andquantitative detection of a chemical, biochemical or biological speciesin a sample, which comprises: (i) contacting the surface of an articleas claimed in claim 70 with the sample; and (ii) measuring the change insurface plasmon resonance of the surface of said article as a result ofthe binding of the chemical, biochemical or biological species onto saidsurface whereby a quantitative measure of the species being assayed isprovided.